Rag GTPases mediate amino acid-dependent recruitment of TFEB and MITF to lysosomes

Abstract

The mTORC1 complex supports cell growth and proliferation in response to energy levels, growth factors, and nutrients. The Rag guanosine triphosphatases (GTPases) activate mTORC1 in response to amino acids by promoting its redistribution to lysosomes. In this paper, we identify a novel role for Rags in controlling activation of transcription factor EB (TFEB), a master regulator of autophagic and lysosomal gene expression. Interaction of TFEB with active Rag heterodimers promoted recruitment of TFEB to lysosomes, leading to mTORC1-dependent phosphorylation and inhibition of TFEB. The interaction of TFEB with Rags required the first 30 residues of TFEB and the switch regions of the Rags G domain. Depletion or inactivation of Rags prevented recruitment of TFEB to lysosomes, whereas expression of active Rags induced association of TFEB with lysosomal membranes. Finally, Rag GTPases bound and regulated activation of microphthalmia-associated transcription factor, suggesting a broader role for Rags in the control of gene expression. Our work provides new insight into the molecular mechanisms that link nutrient availability and TFEB localization and activation.

Figure 1.

Rag GTPases are required for recruitment of TFEB to lysosomes. (A and B) ARPE-19 cells were transfected with siRNA duplexes to raptor, RagA+B, p18, or nontarget. 60 h after transfection, cells were infected with adenovirus expressing either TFEB-FLAG-WT (A) or TFEB-FLAG-S211A (B). 12 h later, cells were fixed, permeabilized with 0.2% Triton X-100, and double stained with antibodies against FLAG (used to detect TFEB) and mTOR. Bars, 10 µm. (C) Quantification of A. (D) Quantification of B. Values are means ± SD of three independent experiments. ***, P < 0.001.

Figure 2.

TFEB interacts with active Rag heterodimers. (A) ARPE-19 cells were nucleofected with the indicated Rag-expressing plasmids. 6 h after nucleofection, cells were infected with adenovirus expressing TFEB-FLAG-WT. 12 h later, cells were lysed and subjected to immunoprecipitation with the anti-FLAG antibody. The immunoprecipitates were analyzed by immunoblotting with antibodies against Rag proteins and FLAG (used to detect TFEB-WT). (B) ARPE-19 cells were nucleofected with the indicated Rag- and TFEB-GFP–expressing plasmids. 18 h later, cells were lysed and subjected to immunoprecipitation with the anti-HA antibody (used to immunoprecipitate Rag proteins). The immunoprecipitates were analyzed by immunoblotting with antibodies against GST and GFP (used to detect Rag proteins and TFEB-WT, respectively). (C) ARPE-19 coexpressing TFEB and the indicated Rag-expressing plasmids were lysed and subjected to immunoprecipitation with the anti-FLAG antibody. The immunoprecipitates were analyzed by immunoblotting with antibodies against GST and FLAG (used to detect Rag proteins and TFEB-WT, respectively). (D) Immunoblotting analysis of coimmunoprecipitated TFEB-FLAG-S211A with Rag heterodimers. IP, immunoprecipitation.

Figure 3.

Regulation of endogenous TFEB by mTOR and Rags. (A) The indicated cell lines were incubated in medium containing DMSO or 250 nM Torin-1 for 1 h. Cells were then lysed and subjected to immunoblotting with antibodies against TFEB (used to detect endogenous TFEB) and actin. (B) HeLa cells were treated as indicated in A, and nuclei and membrane plus cytosol fractions were obtained by low speed centrifugation. Proteins from different fractions were subjected to immunoblotting with antibodies against TFEB (used to detect endogenous TFEB), Lamp1, or Histone H3. (C) Immunofluorescence confocal microscopy showing nuclear and lysosomal localization of endogenous TFEB upon treatment of HeLa cells with Torin-1 as indicated in A. (D) HeLa cells were starved in serum- and amino acid–free medium (starvation) for 3 h and analyzed by immunoblotting with antibodies against TFEB. (E) HeLa cells were incubated in normal medium (control) and serum- and amino acid–free medium (Starvation) for 4 h or starved for 4 h followed by restimulation with amino acids (starvation + amino acids) for 30 min and analyzed by immunofluorescence with antibodies against TFEB (endogenous TFEB is shown in green) and Lamp1 (red). The region within the dotted box is magnified in the insets. (F) HeLa cells were nucleofected with the indicated Rag-expressing plasmids. 18 h later, cells were lysed, and RagB/D heterodimers were pulled down using glutathione–Sepharose beads. Proteins bound to the beads were analyzed by immunoblotting with antibodies against TFEB and GST (used to detect endogenous TFEB and Rag proteins, respectively). Bars, 10 µm.

Figure 4.

Inactivation of endogenous Rags by starvation prevents lysosomal localization of TFEB. (A) ARPE-19 cells were infected with adenovirus expressing TFEB-FLAG-WT. 16 h later, cells were incubated with 250 nM Torin-1 for 1 h or starved in serum- and amino acid–free medium for 3 h. Cells were then fixed, permeabilized with 0.2% saponin, and double stained with antibodies against TFEB (used to detect recombinant TFEB) and Lamp1. (B) ARPE-19 cells were infected with adenovirus expressing TFEB-FLAG-S211A. 12 h later, cells were starved in serum- and amino acid–free medium for 4 h (starvation) or kept in complete medium (control). Cells were then fixed, permeabilized with 0.2% Triton X-100, and stained with antibodies against FLAG (used to detect TFEB-S211A). (C) HeLa cells were incubated with 250 nM Torin-1 for 1 h or starved in serum- and amino acid–free medium for 4 h with the addition of Torin-1 during the last hour of starvation. Cells were then washed, fixed, permeabilized with 0.2% Triton X-100, and double stained with antibodies against TFEB (used to detect endogenous TFEB) and Lamp1. Regions within the dotted boxes are magnified in the insets. Bars: (A [main images], B, and C) 10 µm; (A, insets) 5 µm.

Figure 5.

The activation state of Rags determines localization and activation of TFEB. (A and B) ARPE-19 cells were transfected with either active or inactive RagB/D heterodimers and infected with adenovirus expressing TFEB-FLAG-WT (A) or TFEB-FLAG-S211A (B). After 12 h, cells were double stained with antibodies against FLAG and GST (used to detect TFEB or Rag proteins, respectively). (C) Quantification of TFEB-WT nuclear localization from A. (D) Quantification of TFEB-S211A lysosomal localization from B. (E and F) ARPE-19 cells were cotransfected with plasmids expressing TFEB-GFP together with inactive (E) or active Rag heterodimers (F). Cells were then incubated with 250 nM Torin-1 for 1 h (E) or starved in serum- and amino acid–free medium for 3 h (F). Anti-GST antibodies were used to detect Rag proteins. Arrows point to cells that do not express Rag heterodimers. Values are means ± SD of three independent experiments. ***, P < 0.001; *, P < 0.05. Bars, 10 µm.

Figure 6.

The N-terminal region of TFEB is necessary for interaction with Rag heterodimers and lysosomal localization. (A) Summary of the nuclear and lysosomal distribution of several TFEB amino acid and deletion mutants in ARPE-19 cells treated with either DMSO or Torin-1. (B) ARPE-19 cells were nucleofected with the indicated Rag- and TFEB-expressing plasmids. After 12 h, cell lysates were immunoprecipitated with the anti-FLAG antibody and analyzed by immunoblotting with antibodies against FLAG and GST (used to detect TFEB and Rag proteins, respectively). (C) FLAG-tagged TFEB-WT or TFEB deletion mutants were immunoprecipitated with the anti-FLAG antibody and analyzed by immunoblotting with antibodies against FLAG, 14-3-3 binding motif, or 14-3-3. (D) Immunofluorescence confocal microscopy showing the subcellular distribution of TFEB-WT and TFEB-S3A/R4A mutant upon incubation with DMSO (vehicle) or 250 nM Torin-1 for 1 h. Cells were fixed, permeabilized with 0.2% Triton X-100, and stained with antibodies against FLAG (used to detect TFEB). (E) ARPE-19 cells expressing either TFEB-S211A or TFEB-S211A-Δ30 were double stained with antibodies against TFEB and Lamp1. Regions within the dotted boxes are magnified in the insets. IP, immunoprecipitation. Bars, 10 µm.

Figure 7.

The first 30 amino acids of TFEB are sufficient for binding to active Rag heterodimers. (A) ARPE-19 cells coexpressing active RagB/D heterodimer and the indicated TFEB plasmids were fixed, permeabilized with 0.2% saponin, and stained with antibodies against GST (used to detect Rag proteins). Regions within the dotted boxes are magnified in the insets. Bars, 10 µm. (B) ARPE-19 cells were cotransfected with active RagB/D heterodimers and the indicated TFEB constructs. After 18 h, cell lysates were immunoprecipitated with the anti-HA antibody (used to immunoprecipitate Rag proteins) and immunoblotted with antibodies against GFP and GST (used to detect TFEB-GFP and Rag proteins, respectively). The white line indicates that intervening lanes have been spliced out. (C) Immunofluorescence confocal microscopy showing the subcellular distribution of TFEB-(1–30)-GFP in ARPE-19 cells coexpressing active RagB/D heterodimers (antibodies against GST were used to detect Rags). Bar, 4 µm. (D) Relative RT-PCR analysis of the mRNA expression of autophagy (ATG9B and UVRAG) and lysosomal (MCOLN1) genes in ARPE-19 cells infected with the indicated adenovirus for 48 h. The values are expressed as a ratio to RNA from cells infected with control adenovirus (Ad.-Null). Values are means ± SD of two independent experiments. ***, P < 0.001; **, P < 0.01. (E) Cells were infected with adenovirus as in D and analyzed by immunoblotting with the indicated antibodies. IP, immunoprecipitation.

Figure 8.

Rag GTPases and mTORC1 regulate the function of MITF. (A) Multisequence alignment of the first 30 amino acids of TFEB with MITF isoforms 1, 2, 3, and 7. The green arrows indicate the residues identified as essential for the interaction between TFEB and Rag proteins. Blue letters are to indicate conserved amino acid homology between the different proteins. (B–D) ARPE-19 cells were infected with adenovirus expressing MITF1-FLAG. 16 h later, cells were incubated with DMSO or 250 nM Torin-1 for 1 h and analyzed by immunoblotting (C) or immunofluorescence (B and D) with anti-FLAG antibodies. The region within the dotted box is magnified in the insets. Bars: (B and D, main images) 10 µm; (D, insets) 5 µm. (E) ARPE-19 cells were incubated with 250 nM Torin-1 for 1 h. Cells were then lysed and analyzed by immunoblotting with antibodies against MITF. (F) HEK-293T cells were incubated in medium containing DMSO or Torin-1 for 1 h. Cells were lysed, and nuclei and membrane plus cytosol fractions were obtained by low speed centrifugation. Proteins from the different fractions were subjected to immunoblotting with antibodies against MITF, Lamp1, and Histone H3. (G) ARPE-19 cells expressing active (RagB_GTP/RagD_GDP) or inactive (RagB_GDP/RagD_GTP) Rag heterodimers were immunoprecipitated with the anti-FLAG antibody and immunoblotted with antibodies against FLAG and GST (used to detect MITF and Rag proteins, respectively). (H) HEK-293T cells expressing active (RagB_GTP/RagD_GDP) or inactive (RagB_GDP/RagD_GTP) Rag heterodimers were pulled down using glutathione–Sepharose beads and immunoblotted with antibodies against GST and MITF (used to detect Rag proteins and endogenous MITF, respectively). IP, immunoprecipitation.

Figure 9.

Rag GTPases regulate recruitment of MITF and TFEB to lysosomes. (A and B) ARPE-19 cells were nucleofected with the indicated Rag- and MITF-expressing plasmids. 12 h later, cells were fixed, permeabilized with 0.2% Triton X-100, and double stained with antibodies against FLAG and GST (used to detect MITF and Rag proteins, respectively). Bars, 10 µm. (C) Model representing the mechanism of TFEB and MITF regulation by Rag GTPases. (top) In nutrient-rich conditions, active Rags promote recruitment of mTORC1 and TFEB to lysosomes, thus facilitating mTORC1-dependent phosphorylation of TFEB. Phosphorylation of TFEB at S211 creates a binding site for 14-3-3 and results in sequestration of TFEB in the cytosol. We suggest that 14-3-3 may mask the Rag-binding domain in TFEB (represented in yellow). (bottom) In the absence of amino acids, Rag GTPases and mTORC1 are inactivated. Dissociation of the TFEB–14-3-3 complex allows transport of TFEB to the nucleus and TFEB-mediated activation of a transcriptional network that promotes autophagy, lysosomal biogenesis, and increased lysosomal degradation. Our model proposes that some MITF isoforms might be regulated in a similar manner. The interaction of MITF-1 with 14-3-3 has been previously described (Bronisz et al., 2006). Please note that mTORC1-dependent phosphorylation of MITF-1 S280 has not been reported and is merely speculative. The representation of the Ragulator–Rag–mTORC1 complex is based on the recent crystal structured described by Gong et al. (2011). P, phosphorylation.